By the year 2030, cardiovascular diseases (CVD) and chronic kidney disorders (CKD) are predicted to be the highest grossing mortality factors worldwide. Screening for biochemical metabolite markers in populations at high risk for these diseases is crucial from a diagnostic point of view.
Various clinical metabolite biomarkers that are implicated in various diseases also have structural isomers, some of which may or may not be implicated in disease manifestations. A few examples for such structural isomers include leucine (Leu)/isoleucine (Ile), associated with diabetes and obesity, asymmetric dimethyl arginine (ADMA)/symmetric dimethyl arginine (SDMA) implicated in cardiovascular diseases or chronic kidney disorders; methylmalonic acid (MMA)/succinic acid (SA) in methylmalonic acidemia; alanine/sarcosine in prostate cancer and bilirubin/lumirubin in neonatal jaundice therapy.
In cases wherein both the isomers are implicated in disease, the ratio of the isomers has been found to be crucial in determining the fate of certain diseases and can independently determine the mortality and morbidity. DMA ratio, known as ADMA catabolism index has been studied as a marker in critically ill patients for organ failure and sepsis. Similarly, co-protoporphyrin isomer ratios are indicative of variegate and hereditary porphyria. ADMA, an inhibitor of nitric oxide synthase (NOS) is related to CVD whereas SDMA is a marker for glomerular filtration rate (GFR) and serves as an indicator for CKD. The ratio of ADMA to SDMA is known as ADMA catabolism ratio; this has prognostic value as it determines the extent of accumulation of dimethyl arginine in body.
Leu and Ile are essential branched chain amino acids (BCAA) present only in food. BCAA are important as they are metabolized in the muscles and not in the liver, followed by subsequent conversion to acetyl CoA and succinyl CoA, which are eventually taken up by the tri-carboxylic acid cycle. Therefore, Leu and Ile degradation is altered in people suffering from liver disease, diabetes and obesity.
The conversion of methylmalonyl CoA to succinyl CoA, which requires the presence of vitamin B-12 (Vit-B12) is an important biochemical reaction in the degradation pathway of Ile. Deficiency of Vit-B12 leads to accumulation of MMA in blood leading to methylmalonic acidemia. MMA is a specific indicator for Vit-B12 deficiency and is also observed in patients suffering from cardiovascular ailments and neonatal disorders. SA is interference for MMA in chromatographic separations and a structural isomer of MMA.
Significantly, distinguishing and determination of structural isomer pairs of Leu/Ile, MMA/SA and ADMA/SDMA gives insights into intricate disease linkages between CVD, CKD, and diabetes. Other structural isomers that are implicated in diseases are bilirubin/lumirubin; retinoic acid isomers (vitamin-A deficiency); glucose/fructose and alanine/sarcosine. Infantile jaundice is treated with phototherapy where newborns are exposed to different light wavelengths and bilirubin levels are reduced in due course of time. With phototherapy, bilirubin is converted to its different isomers, a few are configurational isomers, whereas, the other one is a structural one, lumirubin. The structural isomer of sarcosine is L-alanine and has been implicated as a biomarker in progression of prostate cancer.
These conversions are considered as indicators for effective treatment of infantile jaundice. The monosaccharide isomers glucose and fructose is another pair of structural isomers. Alterations in enzymatic conversion of fructose to glucose lead to accumulation and subsequent conversion of fructose to fat. In recent years, adulteration of food with high fructose corn syrup has caused concerns as fructose has been linked with obesity and diabetes.
From the environmental point of view, the pesticide 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT) and its structural isomer (2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1,1-trichloroethane) are also routinely screened from environmental matrices.
In view of the above, structural isomer separation and detection is of vital importance with applications in diverse areas such as disease environmental, pesticides, diagnosis, food and nutrition, drug synthesis and development.
However, it is difficult and challenging to detect these isomers from complex biological mixtures using existing analytical techniques and methodologies. Conventional methods employed include chromatography based separation with or without derivitization and studying them using mass spectrometry. Routine chromatographic techniques namely, liquid and gas chromatography coupled with tandem mass spectrometry (MS/MS) are the usual ways of detecting Ile/Leu, MMA/SA, ADMA/SDMA, glucose/fructose. Alternative techniques such as ELISA (ADMA/SDMA), UV (bilirubin/lumirubin) are also used for differentiation of isomers. Alanine/sarcosine is conventionally separated by GC-MS though;
recently differential mobility tandem mass spectrometry was used for their differentiation. Interestingly, the absolute concentration of the isomer pair (together, not the individual isomers) has often been reported in literature. Often biotransformations and/or interconversions from one isomer form to another are missed out and if a method does not take into account the individual isomer contributions or only screens for only one of the isomers. For example, DDT and its environmental degradation are monitored but, not all studies report the altering ratios of the individual DDT isomers subsequently occurring with biodegradation. This aspect of contributions of individual isomers that form a structural pair in clinical context and their biochemical fate are of significant importance. In analytical and pathological laboratories, knowledge of the relative variation of analyte levels, for example in a diseased state as compared to the baseline healthy or normal subjects is important. By extension, in the case of structural isomers, this relative variation of the individual isomers in a pair is important.
The overall approaches that involve absolute or relative concentrations currently in use have the following disadvantages: (a) limiting due to internal standards and isotopes that many times are either not available or may not be optimized when used with mass spectrometry based quantitation (b) cumbersome with multiple steps, and requiring different set of calibrators for different concentration ranges and (c) may propagate systemic errors affecting the quantitation.
Additionally, these methods also suffer from lack of throughput needed for the analysis of large numbers of samples; require either an internal standard or isotope labelled standard for quantitation.
Isomer detection and quantitation using mass spectrometry has been used previously especially along with chromatographic techniques that aid in isomer separation prior to mass analysis. A few reports and patents are also available, though most of the methods have been developed using triple/quadruple instruments equipped with single/multiple reaction monitoring platforms.
Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is a chromatographic separation free method and at the same time enables high throughput processing of samples. Label free mass spectrometry methods reduce the cost of analysis and bypasses synthesis for a particular isotope label incorporated chemical entity. It is challenging to synthesize chemical structures containing labels and practically impossible for untargeted, unknown and unpredicted structures that usually span the entire mass spectrum range.
Any quantitative method requires normalization of data; in mass spectrometry exogenous internal standards serve that purpose. Tandem mass spectrometry relies on reference standards generally labelled with stable isotopes for that purpose. However, it is possible to achieve normalization without using an exogenous internal standard.
In a Perkin Elmer patent having WIPO Publication No. WO/2007/103124 disclose a method of differentiation of ADMA and SDMA in a mixture using electrospray ionization tandem mass spectrometry (ESI-MS/MS), however, the method uses an isotopic label as an internal standard for quantitation.
Furthermore, handling and processing all the data generated by analytical techniques requires software that gives the required output based on peak area/intensity. This removes any human errors and increases the throughput of the analysis significantly. In some cases the data processing could involve multiple steps that are difficult and time consuming if performed manually without using an algorithm. Any clinical diagnostic application needs throughput. MALDI MS/MS offers high throughput along with sensitive analysis. However, any high throughput technique generates large volumes of data that can only be managed using algorithms. Thus, high throughput platforms such as MALDI/MS estimating structural isomers from mixtures used for high throughput analysis implies generation of large amount of data alongside and thus, software that can process, distinguish, model, quantify and estimate diagnostic values holds clinical significance.
Bearing in mind the above requirements for quick analysis, the instant inventors have used an in-house developed algorithm, which can quantify isomers identified based on their exact masses' using the respective peak heights or areas. The instant method does not require chromatographic separation, significantly reduces the analysis time and greatly simplifies the analysis. Thus, analytical methods, processes and algorithms developed to work with the method to estimate isomers make them more user friendly and enable large scale implementation for societal use and wider access of diagnostic tools.
In the light of the foregoing there is a need in the art to develop a method for identification and quantification of the structural isomers in a mixture that is devoid of the requirements of isotopic labeling, internal standards, and chromatographic separations.
The focus of the instant invention pertains to isotopic label and internal standard free, chromatography free quantitative analysis of tandem mass spectra obtained for structural isomers and interpreted/processed by an algorithm developed in-house. And applicability of methods using these for diverse sets of end applications whose use includes mass spectrometry, cover multiple chemical entities and their varying concentrations (spanning a range of milli grams to femto grams in a unit volume) in a given sample.